PROJECT SUMMARY Decellularized organs hold tremendous promise as scaffolds for the engineering of large transplantable grafts. Achieving this vision will require a method to reconstruct functional vascular trees within such scaffolds. The objective of this pilot R03 study is to apply the lessons we have learned from vascularizing microfluidic collagen- and fibrin-based gels, towards the vascularization of decellularized scaffolds. Our prior studies point toward the dominance of physical signals in promoting functional vascularization. Surprisingly, physical signals have rarely been examined as potential promoters of vascularization in decellularized scaffolds, and we believe they may be the missing ingredient needed to obtain durable, functional vascular networks. The proposed work will use in vitro models of decellularized capillaries and microvessels to quickly screen vascularization conditions, with previously identified physical signals as a starting point. The most promising conditions identified in these screens will be tested on decellularized rat fat pads and lungs ex vivo. Physiological and histological analyses, with emphasis on integrity on the endothelial barrier, will provide organ-level and microscale measures of vascular function. Vascularized scaffolds will be implanted orthotopically in the rat to determine the functionality of the vessels in vivo. At completion, the proposed work will yield proof-of-principle revascularized organ-scale scaffolds that are ready to accept parenchymal cell grafts, for the eventual generation of vascularized functional organs.